Starch consists of two main polysaccharides, amylose and amylopectin. Amylose is a linear polymer containing α-1,4 linked glucose units, while amylopectin is a highly branched polymer consisting of a α-1,4 linked glucan backbone with α-1,6 linked glucan branches. In most plant storage reserves amylopectin consitutes about 75% of the starch content. Amylopectin is synthesized by the concerted action of soluble starch synthase and starch branching enzyme [α-1,4 glucan: α-1,4 glucan 6-glycosyltransferase. EC 2.4.1.18]. Starch branching enzyme (SBE) hydrolyses α-1,4 linkages and rejoins the cleaved glucan, via an α-1,6 linkage, to an acceptor chain to produce a branched structure. The physical properties of starch are strongly affected by the relative abundance of amylose and amylopectin, and SBE is therefore a crucial enzyme in determining both the quality and quality of starches produced in plant systems.
Starches are commercially available from several plant sources including, maize, potato and cassava. Each of these starches has unique physical characteristics and properties and a variety of possible industrial uses. In maize there are a number of naturally occurring mutants which have altered starch composition such as high amylopectin types (“waxy” starches) or high amylose starches but in potato and cassava no such mutants exist on a commercial basis as yet.
Genetic modification offers the possibility of obtaining new starches which may have novel and potentially useful characteristics. Most of the work to date has involved potato plants because they are amenable to genetic manipulation i.e. they can be transformed using Agrobacterium and regenerated easily from tissue culture. In addition many of the genes involved in starch biosynthesis have been cloned from potato and thus are available as targets for genetic manipulation, for example, by antisense inhibition of expression or sense suppression.
Cassava (Manihot esculenta L. Crantz) is an important crop in the tropics, where its starch-filled roots are used both as a food source and increasingly as a source of starch. Cassava is a high yielding perennial crop that can grow on poor soils and is also tolerant of drought. Cassava starch being a root-derived starch has properties similar but not identical to potato starch and is composed of 20-25% amylose and 75-80% amylopectin (Rickard et al., 1991. Trop. Sci. 31, 189-207). Some of the genes involved in starch biosynthesis have been cloned from cassava, including starch branching enzyme I (SBE I) (Salehuzzaman et al., 1994 Plant Science 98, 53-62), and granule bound starch synthase I (GBSS I) (Salehuzzaman et al., 1993 Plant Molecular Biology 23, 947-962) and some work has been done on their expression patterns although only in in vitro grown plants (Salehuzzaman et al., 1994 Plant Science 98, 53-62).
In most plants studied to date e.g. maize (Boyer & Preiss. 1978 Biochem. Biophys. Res. Comm. 80, 169-175), rice (Smyth. 1988 Plant Sci. 57, 1-8) and pea (Smith, Planta 175, 270-279), two forms of SBE have been identified, each encoded by a separate gene. A recent review by Burton et al., (1995 The Plant Journal 7, 3-15) has demonstrated that the two forms of SBE constitute distinct classes of the enzyme such that, in general, enzymes of the same class from different plants may exhibit greater similarity than enzymes of different classes from the same plant. In their review, Burton et al. termed the two respective enzyme families class “A” and class “B”, and the reader is referred thereto (and to the references cited therein) for a detailed discussion of the distinctions between the two classes. One general distinction of note would appear to be the presence, in class A SBE molecules, of a flexible N-terminal domain, which is not found in class B molecules. The distinctions noted by Burton et al. are relied on herein to define class A and class B SBE molecules, which terms are to be interpreted accordingly.
Many organisations have interests in obtaining modified Cassava starches by means of genetic modification. This is impossible to achieve however, unless the plant is amenable to transformation and regeneration, and the starch biosynthesis genes which are to be targeted for modification must be cloned. The production of transgenic cassava plants has only recently been demonstrated (Taylor et al., 1996 Nature Biotechnology 14, 726-730; Schöpke et al., 1996 Nature Biotechnology 14, 731-735; and Li et al., 1996 Nature Biotechnology 14, 736-740). The present invention concerns the identification, cloning and sequencing of a starch biosynthetic gene from Cassava, suitable as a target for genetic manipulation.